A. Field of the Invention
The present invention relates to novel proteins and peptides from the putative receptor binding region of human Growth Arrest Specific Gene 6 (Gas6) and to antibodies, including specified portions or variants, specific for at least one such Gas6 peptide or fragment thereof, as well as nucleic acids encoding such anti-Gas6 antibodies, complementary nucleic acids, vectors, host cells, and methods of making and using thereof, including therapeutic formulations, administration and devices.
B. Related Art
Gas6 was first identified among a set of genes that are highly expressed during serum starvation of NIH 3T3 cells [1]. It was later classified as a new member of a vitamin K-dependent protein family closely related to the coagulation factor Protein S [2] [U.S. Pat. No. 5,538,861]. Gas6 and Protein S share common structure features with ˜40% sequence homology. Both proteins contain a γ-carboxyglutamic acid-rich domain (Gla domain) at the N-terminus followed by four Epidermal Growth Factor-like (EGF) repeats and a tandem globular domain (G domain). However, Gas6 lacks the thrombin recognition sites that are important for the coagulation activity of Protein S. The G domain of Gas6 belongs to a superfamily of proteins including basement membrane proteins laminin and agrin and human steroid hormone-binding globulin (or androgen-binding protein) [3].
Gas6 is a putative ligand for the Axl family of receptor tyrosine kinases including Axl (Ark, Ufo, Tro7) [4, 5, and U.S. Pat. No. 5,538,861], Mer (Eyk, Nyk) [6] and Sky (Rse, Tyro3, DtK, Brt, Tif) [7, 8]. Gas6 binds to the receptors with nanomolar affinity and causes receptor auto-phosphorylation. The ability of Gas6 to bind to and activate the receptors requires vitamin K-dependent γ-carboxylation [9, 10]. However, truncated Gas6 or a splice variant of Gas6 containing the G domain is sufficient to activate the Rse receptor [11, 12] [U.S. Pat. No. 6,211,142]. It seems that the G domain is masked without γ-carboxylation. This “mask” can be removed by a properly modified Gla domain or removal of sequence upstream of the G domain. Gas6 is often found to be associated with the cell membrane due to Gla-mediated calcium-dependent binding to membrane phospholipids [13]. In most systems, secreted Gas6 or soluble recombinant Gas6 has full biological activity, although it is unknown if Gas6 is anchored to the membrane during receptor activation. One exception is that 3T3 cells transfected with Gas6, but not soluble Gas6, support growth of hematopoietic progenitor cells [14]. The hematopoietic effect of the transfected cells is not dependent on vitamin K and the nature of membrane association of Gas6 and the mechanism of action is unexplained.
Gas6 is expressed in the lung, intestine and terminally differentiated cells of most organs including capillary endothelial cells, vascular smooth muscle cells (VSMC) and neurons [2, 15]. It is also found in the alpha granules of platelets that are secreted or transported to the cell surface upon activation [16, 17]. Gas6 is not detected in plasma, macrophages, basophils, neutrophils, or lymphocytes. Under pathological conditions, Gas6 is up regulated at sites of inflammation, vessel injury, and in VSMCs of atherosclerotic plaques [18-20]. Axl is expressed by vascular endothelial cells, CD34+progenitor cells, bone marrow stromal cells, monocytes and macrophages, but not in granulocytes or lymphocytes [5, 21-23]. Neuronal cells and many peripheral cells also express Axl. Mer is expressed in lung, kidney, ovary, prostate, mononuclear cells, monocytes, and macrophages, but not in granulocytes or peripheral blood B or T cells [24, 25]. While Sky is mainly expressed in adult brain, it is also found in gonadal tissues, kidney, and in regions of lymphoid tissues that exclude B or T cells [26, 27]. All three receptors are found in human platelets [17].
Over the past decade, Gas6 has been implicated in many cellular functions such as cell growth, apoptosis, cell adhesion and migration, phagocytosis, and possibly hematopoiesis. Gas6 is a potent mitogen for human Schwann cells [28]. It has been disclosed that Gas6 can be used to support cultured Schwann cells for the treatment of neuronal injury [U.S. Pat. No. 5,721,139]. Purified Gas6 also induces proliferation of serum-depleted NIH 3T3 cells [29] and density-inhibited C57 mammary cells [30]. Mesangial cell proliferation, a hallmark of glomerular sclerosis, can be stimulated by Gas6 [31]. In a model of glomerulonephritis induced by Thy 1.1 antibody, Gas6 and Axl levels are dramatically increased in mesangial cells. Injection of wafarin, an inhibitor of γ-carboxylation, or an extracellular portion of Axl suppresses mesangial cell proliferation in this disease model, suggesting that Gas6 plays a role in glomerular disease. Proliferation of VSMC and mesangial cells is also a feature of chronic rejection after kidney transplant. Moreover, Gas6 and Sky are highly expressed in normal kidney and the level of Gas6 is significantly higher in kidney tissues of allografts and isografts [32]. Thus, it is possible that Gas6 contributes to the pathogenesis of chronic rejection. In addition, Gas6 can stimulate GnRH neuronal cell migration [33], suggesting a role of Gas6 in the central nervous system.
Under certain conditions, Gas6 prevents cell death. Gas6 rescues human umbilical vein endothelial cells from apoptosis induced by serum-deprivation or tumor necrosis factor α [18]. Gas6 is also a survival factor for several other types of cells under serum deprivation, including NIH 3T3 fibroblast cells [29, 34, 35], cancer cells [36, 37], Gonadoptropin-releasing hormone (GnRH) neuronal cells [38] and hippocampal neuronal cells [39]. In contrast, endothelial cells from Gas6 knockout mice are protected from cell death induced by cytokines and anti-Fas antibodies [WO Patent No. 00/76309]. Also, the lack of Gas6 in mice suppresses angiogenesis induced by VEGF-matrigel. Since the ability of activated endothelial cells to secrete cytokines and growth factors has been greatly compromised in Gas6 knockout mice, Gas6 may contribute to endothelial cell apoptosis and angiogenic by regulating local levels of cytokines and growth factors.
The Axl family of receptors is present in many different types of tumor cells and is implicated in neoplasia. Axl, cloned from patients with chronic myelogenous leukemia (CMA), was the first of the family to be identified [Liu, 1988 #205] [40]. Elevated levels of Axl are associated with metastatic colon cancer and lunch cancer [41, 42]. Although none of the receptors or Gas6 are expressed in B and T cells [27], both Axl and Sky are expressed in myeloid leukemic blasts, while Mer is found in neoplastic T- and B-cell lines [21, 24, 43]. Mammary tumors but not non-tumorigenic progenitors express elevated levels of Sky [44]. Axl, Mer, and Sky are all capable of inducing transformation of fibroblasts [40, 45, 46]. Although a high level of Gas6 is found in multiple myeloma [47], the potential neoplastic effects of the receptors may be mediated by other ligand(s) as well. The most convincing evidence for other ligands for these receptors comes from studies of receptor and Gas6 knockout mice. The phenotype in the receptor triple knockout mice is more severe than that in Gas6 knockouts. Mice lacking all three receptors have multiple organ failure, adult blindness, lack of sperm in males and develop severe autoimmunity [27, 48]. Gas6 knockout mice do not show any obvious phenotype unless challenged under pathological conditions such as thrombosis and endothelial activation [17] [WO Patent No. 00/76309]. Given the similarity of Gas6 to Protein S, it is possible that protein S may have a role in activation of Axl, Mer, and Sky under certain physiological conditions. Protein S appears to bind to Sky in vitro. However, bovine and human Protein S bind well to murine Sky but not to their homologous receptor [4, 7].
Through interaction with Mer, Gas6 seems to play a role in outer segment phagocytosis by retinal pigment epithelial cells. Vertebrate photoreceptors undergo daily phagocytosis of photoreceptor outer segments by the adjacent retinal pigment epithelium (RPE). The Royal College of Surgeons (RCS) rat, with inherited homozygous deletion of Mer, suffers from retinal degeneration due to an inability of RPE cells to clear the outer segment [49]. Phagocytosis of outer segment by cultured rat RPE cells can be stimulated by Gas6 [50]. Mer is also reported to play a role in phagocytosis of apoptotic cells by macrophages, an important process to prevent inflammation and autoimmunity against intracellular antigens [51]. Mutant mice expressing kinase-deleted Mer have increased autoantibodies, and macrophages from the mutant fail to clear apoptotic thymocytes. The phenotype of autoimmune response is more severe in the receptor triple knockout [27]. It is unclear whether Gas6 mediates any of these latter responses through Mer since Gas6 knockout mice are not reported to develop autoimmunity.
Gas6 is expressed in hematopoetic tissues and seems to regulate erythropoiesis under pathological condition [52, 53]. Gas6 knockout mice have a reduced erythrocyte count and fewer cells of erythroid lineage in bone marrow, spleen, and fetal liver. The hematocrit in the blood is, however, normal in the mutant mice. Gas6 deficient mice are more susceptible to acute hemolytic anemia induced by phenylhydrazine or autoimmune hemolytic anemia induced by NZB-derived 4C8 IgG2a anti-red cell antibody. Even though bone marrow erythroid precursors do not express Gas6, the erythropoietic effect of Gas6 may be mediated through Sky as it is found in erythroid precursors.
Vascular neointima formation, a process involving VSMC proliferation and migration, contributes to the formation and progression of lesions of restenosis and atherosclerosis. Treatment of VSMC with Gas6 causes enhancement of the growth response to thrombin and angiotensin II [54], induction of cell migration [55] and prevention of serum-deprivation-induced cell death [56]. Upon balloon injury, the level of Gas6 and Axl rises dramatically in the rat carotid artery [19]. This up-regulation of Gas6 and Axl parallels the time course of migration of the VSMC from media to intima, suggesting a role of Gas6 in the pathogenesis of restenosis and atherosclerosis. In fact, arterial stenosis induced by carotid artery ligation is reduced in Gas6 deficient mice [WO Patent No. 00/76309].
One of the common features of all cardiovascular disorders is activation of endothelium, which induces inflammatory responses causing severe damage in affected tissues. Gas6 may be involved directly or indirectly in the inflammatory response. A role of Gas6 in leukocyte adhesion during inflammatory response is controversial. In murine myeloid progentitor 32D cells, Gas6 promotes Axl-mediated cell adhesion [57]. A high concentration of Gas6, on the other hand, inhibits granulocyte adhesion to endothelial cells [58]. Interestingly, endothelial cells lacking Gas6 fail to induce expression of cytokines, adhesion molecules and tissue factor upon TNFα or endotoxin stimulation [WO Patent No. 00/76309], indicating a pro-inflammatory role of Gas6. Leukocyte adhesion to the arterial wall upon endotoxin challenge is markedly reduced in Gas6 deficient mice. In an ischemic stroke model, the infarction size in the Gas6 knockout mice is significantly reduced, possibly as a result of suppression of the inflammatory response [WO Patent No. 00/76309].
Studies from Gas6 deficient mice have revealed a surprising function of Gas6 in thrombosis [17]. Platelet aggregation and secretion stimulated by other aganonists are impaired in Gas6 knockout mice. Platelet aggregates induced by thrombin from the mutant are loosely packed, suggesting the possibility that lack of Gas6 might prevent formation of stable platelet plaque in vivo. Indeed, Gas6 contributes to thrombus generation in vivo. Gas6 mutant mice or wild type mice treated with neutralizing polyclonal antibodies are protected from lethal challenge of pulmonary thrombosis, a platelet dependent thrombosis model. Gas6 may also contribute to fibrin dependent thrombus formation due to its effect on tissue factor expression in endothelial cells. This is supported by the result from other thrombosis models in which the role of platelet is less prominent [17]. The thrombus size in Gas6 mutant mice is 60-85% smaller than wild type after carotid artery injury- or ligation of inferior vena cava.
There is a controversial role of Gas6 in diabetes, particularly in the development of noninsulin-dependent diabetes mellitus (NIDDM) and insulin-resistant disorders. Transgenic mice ectopically expressing Axl or the extracellular domain of Axl in myeloid cells develop phenotypes similar to NIDDM [59]. These animals display hyperglycemia, hyperinsulinemia, severe insulin resistance, progressive obesity, hepatic lipidsis and pancreatic islet dysplasia, but do not exhibit hyperphagia. These animals express an elevated level of TNFα in serum, which may cause insulin resistance in these mice. Addition of Gas6 to blood samples eliminates LPS-induced TNFα induction. Transgenic mice systemically expressing Gas6, on the other hand, do not show diabetic phenotype. In a different set of experiments, administration of Gas6 in combination with insulin causes higher insulin level than mice treated with insulin alone [WO Patent No. 99/49894]. Further study is required to support a role of Gas6 in diabetes under physiological condition.
Gas6 and Sky are expressed in osteoclasts and seem to be involved in osteoclastic bone resorption [60]. Treatment with Gas6 doubles the amount of pit area on a denite slice resorbed by osteoclast cells. Coincidentally, the level of Gas6 is up regulated in ovariectomized mice receiving estrogen. Ovariectomized mice is a model of postmenopausal osteoporosis caused by estrogen withdrawal. Osteoclast bone resorption is also observed in rheumatoid arthritis and ostoarthritis, which is accompanied by an elevated level of Gas6 [18]. Thus, it is possible that Gas6 contributes to bone loss in patients suffering from osteoporosis and arthritis.
Overall, Gas6 plays an important role in multiple patho-physiological processes, many of which lead to life threatening diseases. Development of Gas6 antibodies will be useful for a variety of diagnostic applications and a broad spectrum of therapeutic applications. In particular, neutralizing monoclonal antibodies or antagonists of human Gas6 can be applied to prevent or treat thromboembolic disease or thrombotic pathologic condition such as ischemic disease (ischemic stroke, ischemic cerebral infarction, acute myocardial infarction, and chronic ischemic heart disease), venous thromboembolism, arterial or venous thrombosis, pulmonary embolism, restenosis following coronary artery bypass surgery or following percutaneous transluminal angioplasty of a coronary artery, diabetic angiopathy and allograft arteriosclerosis. Gas6 antagonists may also be beneficial for preventing or treating other disease conditions such as cancer, atherosclerosis, sepsis, glomerular sclerosis, diabetes, rheumatoid arthritis, osteoarthritis and osteoporosis.
The only reported antibodies to Gas6 are polyclonal preparations, primarily generated by immunizing animals with purified native protein or recombinant full-length protein. Two polyclonal antibodies marketed by Santa Cruz Biotechnology, Inc. are from animals immunized with undisclosed peptide sequences derived from the N- and C-termini of the human Gas6 protein. These disclosed antibodies are not suitable as therapeutic agents and unproven for diagnostic applications.
Accordingly, there is a need for a novel method for generating anti-Gas6 antibodies (also termed Gas6 antibodies) based on structural information for the Gas6 protein.